The invention disclosed in this patent document relates to transmembrane receptors, more particularly to endogenous, constitutively active G protein-coupled receptors for which the endogenous ligand is unknown, and most particularly to the use of such receptors for the direct identification of candidate compounds via screening as agonists, partial agonists or inverse agonists to such receptors.
G protein-coupled receptors share a common structural motif. All these receptors have seven sequences of between 22 to 24 hydrophobic amino acids that form seven alpha helices, each of which spans the membrane. The transmembrane helices are joined by strands of amino acids having a larger loop between the fourth and fifth transmembrane helix on the extracellular side of the membrane. Another larger loop, composed primarily of hydrophilic amino acids, joins transmembrane helices five and six on the intracellular side of the membrane. The carboxy terminus of the receptor lies intracellularly with the amino terminus in the extracellular space. It is thought that the loop joining helices five and six, as well as the carboxy terminus, interact with the G protein. Currently, Gq, Gs, Gi, and Go are G proteins that have been identified. The general structure of G protein-coupled receptors is shown in FIG. 1.
Under physiological conditions, G protein-coupled receptors exist in the cell membrane in equilibrium between two different states or conformations: an xe2x80x9cinactivexe2x80x9d state and an xe2x80x9cactivexe2x80x9d state. As shown schematically in FIG. 2, a receptor in an inactive state is unable to link to the intracellular transduction pathway to produce a biological response. Changing the receptor conformation to the active state allows linkage to the transduction pathway and produces a biological response.
A receptor may be stabilized in an active state by an endogenous ligand or an exogenous agonist ligand. Recent discoveries such as, including but not exclusively limited to, modifications to the amino acid sequence of the receptor provide means other than ligands to stabilize the active state conformation. These means effectively stabilize the receptor in an active state by simulating the effect of a ligand binding to the receptor. Stabilization by such ligand-independent means is termed xe2x80x9cconstitutive receptor activation.xe2x80x9d A receptor for which the endogenous ligand is unknown or not identified is referred to as an xe2x80x9corphan receptor.xe2x80x9d
Generally, the use of an orphan receptor for screening purposes to identify compounds that modulate a biological response associated with such receptor has not been possible. This is because the traditional xe2x80x9cdogmaxe2x80x9d regarding screening of compounds mandates that the ligand for the receptor be known, whereby compounds that competitively bind with the receptor, i.e., by interfering or blocking the binding of the natural ligand with the receptor, are selected. By definition, then, this approach has no applicability with respect to orphan receptors. Thus, by adhering to this dogmatic approach to the discovery of therapeutics, the art, in essence, has taught and has been taught to forsake the use of orphan receptors unless and until the natural ligand for the receptor is discovered. The pursuit of an endogenous ligand for an orphan receptor can take several years and cost millions of dollars.
Furthermore, and given that there are an estimated 2,000 G protein-coupled receptors in the human genome, the majority of which being orphan receptors, the traditional dogma castigates a creative approach to the discovery of therapeutics to these receptors.
GPR3 is a 330 amino acid G protein coupled receptor for which the endogenous ligand is unknown. (Marchese, A. et al. (1994) Genomics 23:609; see also, Iismaa, T. P. et al (1994) Genomics 24:391; see FIG. 1 for reported nucleic acid and amino acid sequence.) GPR3 is constitutively active in its endogenous form. (Eggerick, D. et al. (1995) Biochem. J. 389:837). GPR12 is a 334 amino acid homolog of GPR3; the endogenous ligand for GPR12 is unknown (Song, Z.-H., et al (1995) Genomics, 28:347; see FIG. 1 for reported amino acid sequence). GPR6 is a 362 amino acid homolog of GPR3; the endogenous ligand for GPR6 is unknown (Song, Z.-H. et al, supra.; see FIG. 1 for reported amino acid sequence). GPR6 transcripts are reported to be abundant in the human putamen and to a lesser extent in the frontal cortex, hippocampus, and hypothalamus (Heiber, M. et al. DNA and Cell Biology (1995) 14(1):25; see FIG. 1 for reported nucleic acid and amino acid sequences for GPR6). GPR4 has also been identified as an orphan GPCR (Heiber, M. et al, 14 DNA Cell Biol. 25 (1995)). OGR1, an orphan GPCR, is reported to have a high level of homology with GPR4 (Xu, Y. and Casey, G., 35 Genomics 397 (1996)). GPR21 is a 349 amino acid G protein coupled receptor for which the endogenous ligand is unknown (see GenBank Accession #U66580 for nucleic acid and deduced amino acid sequence). GPR21 has been reported to be located at chromosome 9q33. O""Dowd B. et al., 187 Gene 75 (1997). AL022171 is a human DNA sequence from clone 384F21 on chromosome 1q24. AL022171 has been identified to contain an open reading frame of 1,086 bp encoding for a 361 amino acid protein. (see GenBank Accession number AL022171). AL022171 is 68% homologous to GPR21 (see FIG. 5B). GHSR is also identified as an orphan GPCR (Howard, A. D. et al, 273 Science 974 (1996)).
Disclosed herein are methods for screening of candidate compounds against endogenous, constitutively activated G protein-coupled orphan receptors (GPCRs) for the direct identification of candidate compounds as agonists, inverse agonists or partial agonists to such receptors. For such screening purposes, it is preferred that an endogenous, constitutively activated orphan GPCR:G proteinxe2x80x94fusion protein be utilized.